Lyme disease is the most reported vector-borne disease in the United States. The causative agent, Borrelia burgdorferi (Bb), is an obligate pathogenic spirochete that is transmitted via a tick bite. How Bb is maintained in its enzootic cycle between mammals and ticks is poorly understood; yet, this understanding is critical for developing innovative approaches to disrupt the tick-mammal-tick natural cycle. We and others have identified a two-component signal transduction system, Hk1-Rrp1, that is essential for Bb survival in the tick. Rrp1is a response regulator possessing diguanylate cyclase (DGC) activity that produces cyclic dimeric GMP (c-di-GMP), a new bacterial second messenger that controls the switch between the motile, single-cellular lifestyle and the sessile, multicellular lifestyle (biofilms). We discovered that c-di-GMP-mediated signaling controls a catabolic switch from glucose to glycerol upon the transition from the mammal to the tick, by regulating the transcription of the glp operon for glycerol transport and utilization which are critical for spirochete survival in ticks. Further, we found that the c-di-GMP-binding effector protein PlzA, previously known to influence Bb motility, also controls glp expression as well as synthesis of multiple virulence factors. We hypothesize that, because of the small genome and streamlined regulatory repertoire, Bb employs the same c-di-GMP effector protein, PlzA, or a protein that interacts with PlzA, to coordinate diverse processes (motility, catabolism, virulence) that are involved in the mammal-to-tick transition. To test this hypothesis and to to understand the role of c-di-GMP signaling in the mammal-to-arthropod host transition, we have formulated the following Specific Aims: (1) Elucidate the mechanism of regulation of the glycerol metabolism and virulence by the c-di-GMP- binding protein PlzA in Bb; (2) Identify and characterize new c-di-GMP-binding effector proteins in Bb and interrogate their role in survival in the tick. Outcomes will uncover novel processes underlying the mammal-to-arthropod host transition, novel c-di-GMP effector proteins and new c-di-GMP signaling paradigm.